Bryan A. Nerger

Type I collagen self-assembles into three-dimensional (3D) fibrous networks. These dynamic viscoelastic materials can be remodeled in response to mechanical and chemical signals to form anisotropic networks, the structure of which influences tissue development, homeostasis, and disease progression. Conventional approaches for fabricating anisotropic networks of type I collagen are often limited to unidirectional fiber alignment over small areas. Here, we describe a new approach for engineering cell-laden networks of aligned type I collagen fibers using 3D microextrusion printing of a collagen-Matrigel ink. We demonstrate hierarchical control of 3D-printed collagen with the ability to spatially pattern collagen fiber alignment and geometry. Our data suggest that collagen alignment results from a combination of molecular crowding in the ink and shear and extensional flows present during 3D printing. We demonstrate that human breast cancer cells cultured on 3D-printed collagen constructs orient along the direction of collagen fiber alignment. We also demonstrate the ability to simultaneously bioprint epithelial cell clusters and control the alignment and geometry of collagen fibers surrounding cells in the bioink. The resulting cell-laden constructs consist of epithelial cell clusters fully embedded in aligned networks of collagen fibers. Such 3D-printed constructs can be used for studies of developmental biology, tissue engineering, and regenerative medicine.

Reciprocal epithelial-mesenchymal signaling is essential for morphogenesis, including branching of the lung. In the mouse, mesenchymal cells differentiate into airway smooth muscle that wraps around epithelial branches, but this contractile tissue is absent from the early avian lung. Here, we have found that branching morphogenesis in the embryonic chicken lung requires extracellular matrix (ECM) remodeling driven by reciprocal interactions between the epithelium and mesenchyme. Before branching, the basement membrane wraps the airway epithelium as a spatially uniform sheath. After branch initiation, however, the basement membrane thins at branch tips; this remodeling requires mesenchymal expression of matrix metalloproteinase 2, which is necessary for branch extension but for not branch initiation. As branches extend, tenascin C (TNC) accumulates in the mesenchyme several cell diameters away from the epithelium. Despite its pattern of accumulation, TNC is expressed exclusively by epithelial cells. Branch extension coincides with deformation of adjacent mesenchymal cells, which correlates with an increase in mesenchymal fluidity at branch tips that may transport TNC away from the epithelium. These data reveal novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branching morphogenesis.

Stem cell-derived kidney organoids contain nephron segments that recapitulate morphological and functional aspects of the human kidney. However, directed differentiation protocols for kidney organoids are largely conducted using biochemical signals to control differentiation. Here, the hypothesis that mechanical signals regulate nephrogenesis is investigated in 3D culture by encapsulating kidney organoids within viscoelastic alginate hydrogels with varying rates of stress relaxation. Tubular nephron segments are significantly more convoluted in kidney organoids differentiated in encapsulating hydrogels when compared with those in suspension culture. Hydrogel viscoelasticity regulates the spatial distribution of nephron segments within the differentiating kidney organoids. Consistent with these observations, a particle-based computational model predicts that the extent of deformation of the hydrogel-organoid interface regulates the morphology of nephron segments. Elevated extracellular calcium levels in the culture medium, which can be impacted by the hydrogels, decrease the glomerulus-to-tubule ratio of nephron segments. These findings reveal that hydrogel encapsulation regulates nephron patterning and morphology and suggest that the mechanical microenvironment is an important design variable for kidney regenerative medicine.

When a sessile droplet containing a solute in a volatile solvent evaporates, flow in the droplet can transport and assemble solute particles into complex patterns. Transport in evaporating sessile droplets has largely been examined in solvents that undergo complete evaporation. Here, we demonstrate that flow in evaporating aqueous sessile droplets containing type I collagen-a self-assembling polymer-can be harnessed to engineer hydrated networks of aligned collagen fibers. We find that Marangoni flows direct collagen fiber assembly over millimeter-scale areas in a manner that depends on the rate of self-assembly, the relative humidity of the surrounding environment, and the geometry of the droplet. Skeletal muscle cells that are incorporated into and cultured within these evaporating droplets collectively orient and subsequently differentiate into myotubes in response to aligned networks of collagen. Our findings demonstrate a simple, tunable, and high-throughput approach to engineer aligned fibrillar hydrogels and cell-laden biomimetic materials.